Previous studies have demonstrated that mammary gland development and function was severely impaired in transgenic mice expressing a gain-in-function mutation of the Notch4/Int3 gene from the MMTV viral promoter. Both mammary ductal growth and secretory lobule development was curtailed in these mice. To confirm and extend these findings, mutated Notch4/Int3 was expressed from the whey acidic protein (WAP) promoter whose activity, unlike the MMTV LTR, is restricted to the secretory mammary epithelial population. In transgenic mice carrying the WAP/Int3 construct, mammary ductal growth was unaffected in virgin females, but growth and differentiation of secretory lobules during gestation was profoundly inhibited. Coincidental with the block in lobular secretory differentiation, mammary dysplasia and tumorigenesis occurred in all females by 25 weeks of age. The WAP/Int3 mammary tumors were highly malignant and most tumor-bearing females, irrespective of breeding history developed metastatic lung lesions. These results suggest that WAP-promoter targeted Int3 function is associated with mammary secretory cell differentiation and maintenance in this transgenic model. Consistent with this conclusion, transplants of WAP/Int3 gland into non-transgenic mammary fat pads produced complete mammary ductal outgrowths in virgin FVB/N mice but failed to develop secretory lobules when the females were impregnated. An in vivo transplantation system has been used to evaluate the developmental capacities of specific mouse mammary epithelial cell populations. Specifically, mouse mammary epithelial cells with distinctly limited developmental potentials have been identified using this procedure. Two distinct epithelial cell progenitors have been identified by experiments designed to determine whether basal lobular and ductal phenotypes could develop independently under conditions imposed by a limiting dilution. The prediction that these separate epithelial progenitors must exist was based upon the results from transplantation experiments carried out in epithelium-divested mammary fat pads of syngeneic mice with mammary epithelium from two different transgenic mouse models (WAP-Int3; WAP-TGF-b1). The results demonstrated the following points: 1) lobular, i.e., secretory progenitor cells are present as distinct entities among the mammary epithelial cells found in immature virgin female mice; 2) similarly, ductal epithelial progenitors are present within the same population; 3) lobular progenitors are present in greater numbers, although both cell populations are extremely small; 4) some inocula produced outgrowths with simultaneous development of both lobular and ductal phenotypes. This indicates either cooperative interaction between the two epithelial progenitors or represents evidence for a multipotent epithelial stem cell capable of producing both ductular- and lobular-committed daughters; 5)these findings have important consequences in the design of experiments aimed at developing methodologies for early detection of breast neoplasia, chemopreventive strategies and/or therapeutic approaches to the control of breast malignancy.